First-in-human study of a novel cell death tracer [99mTc]Tc-Duramycin: safety, biodistribution and radiation dosimetry in healthy volunteers.

BACKGROUND: Imaging of cell death can provide an early indication of treatment response in cancer. [99mTc]Tc-Duramycin is a small-peptide SPECT tracer that recognizes both apoptotic and necrotic cells by binding to phosphatidylethanolamine present in the cell membrane. Preclinically, this tracer has shown to have favorable pharmacokinetics and selective tumor accumulation early after the onset of anticancer therapy. In this first-in-human study, we report the safety, biodistribution and internal radiation dosimetry of [99mTc]Tc-Duramycin in healthy human volunteers. RESULTS: Six healthy volunteers (3 males, 3 females) were injected intravenously with [99mTc]Tc-Duramycin (dose: 6 MBq/kg; 473 ± 36 MBq). [99mTc]Tc-Duramycin was well tolerated in all subjects, with no serious adverse events reported. Following injection, a 30-min dynamic planar imaging of the abdomen was performed, and whole-body (WB) planar scans were acquired at 1, 2, 3, 6 and 23 h post-injection (PI), with SPECT acquisitions after each WB scan and one low-dose CT after the first SPECT. In vivo 99mTc activities were determined from semi-quantitative analysis of the images, and time-activity curves were generated. Residence times were calculated from the dynamic and WB planar scans. The mean effective dose was 7.61 ± 0.75 µSv/MBq, with the kidneys receiving the highest absorbed dose (planar analysis: 43.82 ± 4.07 µGy/MBq, SPECT analysis: 19.72 ± 3.42 µGy/MBq), followed by liver and spleen. The median effective dose was 3.61 mSv (range, 2.85-4.14). The tracer cleared slowly from the blood (effective half-life of 2.0 ± 0.4 h) due to high plasma protein binding with < 5% free tracer 3 h PI. Excretion was almost exclusively renal. CONCLUSION: [99mTc]Tc-Duramycin demonstrated acceptable dosimetry (< 5 mSv) and a favorable safety profile. Due to slow blood clearance, optimal target-to-background ratios are expected 5 h PI. These data support the further assessment of [99mTc]Tc-Duramycin for clinical treatment response evaluation. TRIAL REGISTRATION: NCT05177640, Registered April 30, 2021, https://clinicaltrials.gov/study/NCT05177640 .

Preclinical Evaluation of a Novel 18F-Labeled dTCO-Amide Derivative for Bioorthogonal Pretargeted Positron Emission Tomography Imaging.

Pretargeted positron emission tomography (PET) imaging based on the bioorthogonal inverse-electron-demand Diels-Alder reaction between tetrazines (Tz) and trans-cyclooctenes (TCO) has emerged as a promising tool for solid tumor imaging, allowing the use of short-lived radionuclides in immune-PET applications. With this strategy, it became possible to achieve desirable target-to-background ratios and at the same time to decrease the radiation burden to nontargeted tissues because of the fast clearance of small PET probes. Here, we show the synthesis of novel 18F-labeled dTCO-amide probes for pretargeted immuno-PET imaging. The PET probes were evaluated regarding their stability, reactivity toward tetrazine, and pharmacokinetic profile. [ 18 F]MICA-213 showed an extremely fast kinetic rate (10,553 M-1 s-1 in 50:50 MeOH/water), good stability in saline and plasma up to 4 h at 37 °C with no isomerization observed, and the biodistribution in healthy mice revealed a mixed hepatobiliary and renal clearance with no defluorination and low background in other tissues. [ 18 F]MICA-213 was further used for in vivo pretargeted immune-PET imaging carried out in nude mice bearing LS174T colorectal tumors that were previously treated with a tetrazine-modified anti-TAG-72 monoclonal antibody (CC49). Pretargeted µPET imaging results showed clear visualization of the tumor tissue with a significantly higher uptake when compared to the control.

Improved stability of a novel fluorine-18 labeled TCO analogue for pretargeted PET imaging.

INTRODUCTION: Biorthogonal pretargeted imaging using the inverse electron demand Diels Alder (IEDDA) reaction between tetrazine (Tz) and trans-cyclooctene (TCO) is one of the most attractive strategies in molecular imaging. It allows the use of short-lived radioisotopes such as fluorine-18 for imaging of long circulating vectors with improved imaging contrast and reduced radiation dose. Here we aim to develop a novel 18F-labeled trans-cyclooctene (TCO) with improved metabolic stability and assess its potential usefulness in a pretargeted PET imaging approach. METHODS: We have synthetized a new TCO-analogue containing a 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) chelator, allowing radiolabeling by chelation with aluminum fluoride (Al[18F]F). Stability and pharmacokinetic profile of Al[18F]F-NOTA-TCO ([18F]MICA-205) were evaluated in healthy animals at different timepoints after injection of the radiotracer. To assess the potential use of this new PET tracer for tumor targeting, in vivo pretargeted PET imaging was performed in LS174T tumor-bearing mice pre-treated with a tetrazine-modified anti-TAG-72 monoclonal antibody (CC49). RESULTS: The radiotracer was obtained with a radiochemical yield (RCY) of 12.8 ±â€¯2.8% and a radiochemical purity (RCP) of ≥95%. It also showed a promising in vivo stability with 51.9 ±â€¯5.16% of radiotracer remaining intact after 1 h. The biodistribution in healthy mice demonstrated mixed hepatobiliary and renal clearance, with a rapid blood clearance and low uptake in other tissues. The low bone uptake indicated lack of tracer defluorination. Interestingly, a pretargeted PET imaging experiment showed a significantly increased radiotracer uptake (0.67 ±â€¯0.16%ID/g, p < 0.001) in the tumors of mice pre-treated with CC49-tetrazine compared to the CC49 alone (0.16 ±â€¯0.08%ID/g). CONCLUSIONS: [18F]MICA-205 represents a large improvement in in vivo metabolic stability compared to previous reported 18F-labeled TCOs, allowing a clear visualization of tumor tissue in a small-animal pretargeted PET imaging experiment. Despite the favorable in vivo stability and image contrast obtained with [18F]MICA-205, the development of next-generation derivatives with increased absolute tumor uptake is warranted for future pretargeting applications.

Effects of metformin on tumor hypoxia and radiotherapy efficacy: a [18F]HX4 PET imaging study in colorectal cancer xenografts.

BACKGROUND: In a colorectal cancer xenograft model, we investigated the therapeutic effect of metformin on tumor hypoxia with [18F]flortanidazole ([18F]HX4) small-animal positron emission tomography (µPET). We also assessed the additive effect of metformin on long-term radiotherapy outcome and we studied the potential of [18F]HX4 as a predictive and/or prognostic biomarker within this setup. METHODS: Colo205-bearing mice (n = 40) underwent a baseline [18F]HX4 hypoxia µPET/computed tomography (CT) scan. The next day, mice received 100 mg/kg metformin or saline intravenously (n = 20/group) and [18F]HX4 was administered intravenously 30 min later, whereupon a second µPET/CT scan was performed to assess changes in tumor hypoxia. Two days later, mice were further divided into four therapy groups (n = 10/group): control (1), metformin (2), radiotherapy (3), and metformin + radiotherapy, i.e., combination (4). Then, they received a second dose of metformin (groups 2 and 4) or saline (groups 1 and 3), followed by a single radiotherapy dose of 15 Gy (groups 3 and 4) or sham irradiation (groups 1 and 2) 30 min later. Tumor growth was followed three times a week by caliper measurements to assess the therapeutic outcome. RESULTS: [18F]HX4 uptake decreased in metformin-treated tumors with a mean intratumoral reduction in [18F]HX4 tumor-to-background ratio (TBR) from 2.53 ± 0.30 to 2.28 ± 0.26 (p = 0.04), as opposed to saline treatment (2.56 ± 0.39 to 3.08 ± 0.39; p = 0.2). The median tumor doubling time (TDT) was 6, 8, 41, and 43 days in the control, metformin, radiotherapy and combination group, respectively (log-rank p < 0.0001), but no metformin-specific therapy effects could be detected. Baseline [18F]HX4 TBR was a negative prognostic biomarker for TDT (hazard ratio, 2.39; p = 0.02). CONCLUSIONS: Metformin decreased [18F]HX4 uptake of Colo205-tumors, but had no additive effect on radiotherapy efficacy. Nevertheless, [18F]HX4 holds promise as a prognostic imaging biomarker.

Caspase-3 probes for PET imaging of apoptotic tumor response to anticancer therapy.

Apoptosis is a highly regulated process involved in the normal organism development and homeostasis. In the context of anticancer therapy, apoptosis is also studied intensively in an attempt to induce cell death in cancer cells. Caspase activation is a known key event in the apoptotic process. In particular, active caspase-3 and -7 are the common effectors in several apoptotic pathways, therefore effector caspase activation may be a promising biomarker for response evaluation to anticancer therapy. Quantitative imaging of apoptosis in vivo could provide early assessment of therapeutic effectiveness and could also be used in drug development to evaluate the efficacy as well as potential toxicity of novel treatments. Positron Emission Tomography (PET) is a highly sensitive molecular imaging modality that allows non-invasive in vivo imaging of biological processes such as apoptosis by using radiolabeled probes. Here we describe the development and evaluation of fluorine-18-labeled caspase-3 activity-based probes (ABPs) for PET imaging of apoptosis. ABPs were selected by screening of a small library of fluorine-19-labeled DEVD peptides containing different electrophilic warhead groups. An acyloxymethyl ketone was identified with low nanomolar affinity for caspase-3 and was radiolabeled with fluorine-18. The resulting radiotracer, [18F]MICA-302, showed good labeling of active caspase-3 in vitro and favorable pharmacokinetic properties. A µPET imaging experiment in colorectal tumor xenografts demonstrated an increased tumor accumulation of [18F]MICA-302 in drug-treated versus control animals. Therefore, our data suggest this radiotracer may be useful for clinical PET imaging of response to anticancer therapy.

Neuroimaging of Subacute Brain Inflammation and Microstructural Changes Predicts Long-Term Functional Outcome after Experimental Traumatic Brain Injury.

There is currently a lack of prognostic biomarkers to predict the different sequelae following traumatic brain injury (TBI). The present study investigated the hypothesis that subacute neuroinflammation and microstructural changes correlate with chronic TBI deficits. Rats were subjected to controlled cortical impact (CCI) injury, sham surgery, or skin incision (naïve). CCI-injured (n = 18) and sham-operated rats (n = 6) underwent positron emission tomography (PET) imaging with the translocator protein 18 kDa (TSPO) radioligand [18F]PBR111 and diffusion tensor imaging (DTI) in the subacute phase (≤3 weeks post-injury) to quantify inflammation and microstructural alterations. CCI-injured, sham-operated, and naïve rats (n = 8) underwent behavioral testing in the chronic phase (5.5-10 months post-injury): open field and sucrose preference tests, two one-week video-electroencephalogram (vEEG) monitoring periods, pentylenetetrazole (PTZ) seizure susceptibility tests, and a Morris water maze (MWM) test. In vivo imaging revealed pronounced neuroinflammation, decreased fractional anisotropy, and increased diffusivity in perilesional cortex and ipsilesional hippocampus of CCI-injured rats. Behavioral analysis revealed disinhibition, anhedonia, increased seizure susceptibility, and impaired learning in CCI-injured rats. Subacute TSPO expression and changes in DTI metrics significantly correlated with several chronic deficits (Pearson's |r| = 0.50-0.90). Certain specific PET and DTI parameters had good sensitivity and specificity (area under the receiver operator characteristic [ROC] curve = 0.85-1.00) to distinguish between TBI animals with and without particular behavioral deficits. Depending on the investigated behavioral deficit, PET or DTI data alone, or the combination, could very well predict the variability in functional outcome data (adjusted R2 = 0.54-1.00). Taken together, both TSPO PET and DTI seem promising prognostic biomarkers to predict different chronic TBI sequelae.

18F-Flortanidazole Hypoxia PET Holds Promise as a Prognostic and Predictive Imaging Biomarker in a Lung Cancer Xenograft Model Treated with Metformin and Radiotherapy.

Metformin may improve tumor oxygenation and thus radiotherapy response, but imaging biomarkers for selection of suitable patients are still under investigation. First, we assessed the effect of acute metformin administration on non-small cell lung cancer xenograft tumor hypoxia using PET imaging with the hypoxia tracer 18F-flortanidazole. Second, we verified the effect of a single dose of metformin before radiotherapy on long-term treatment outcome. Third, we examined the potential of baseline 18F-flortanidazole as a prognostic or predictive biomarker for treatment response. Methods: A549 tumor-bearing mice underwent a 18F-flortanidazole PET/CT scan to determine baseline tumor hypoxia. The next day, mice received a 100 mg/kg intravenous injection of metformin. 18F-flortanidazole was administered intravenously 30 min later, and a second PET/CT scan was performed to assess changes in tumor hypoxia. Two days later, the mice were divided into 3 therapy groups: controls (group 1), radiotherapy (group 2), and metformin + radiotherapy (group 3). Animals received saline (groups 1-2) or metformin (100 mg/kg; group 3) intravenously, followed by a single radiotherapy dose of 10 Gy (groups 2-3) or sham irradiation (group 1) 30 min later. Tumor growth was monitored triweekly by caliper measurement, and tumor volume relative to baseline was calculated. The tumor doubling time (TDT), that is, the time to reach twice the preirradiation tumor volume, was defined as the endpoint. Results: Thirty minutes after metformin treatment, 18F-flortanidazole demonstrated a significant change in tumor hypoxia, with a mean intratumoral reduction in 18F-flortanidazole tumor-to-background ratio (TBR) from 3.21 ± 0.13 to 2.87 ± 0.13 (P = 0.0001). Overall, relative tumor volume over time differed across treatment groups (P < 0.0001). Similarly, the median TDT was 19, 34, and 52 d in controls, the radiotherapy group, and the metformin + radiotherapy group, respectively (log-rank P < 0.0001). Both baseline 18F-flortanidazole TBR (hazard ratio, 2.0; P = 0.0004) and change from baseline TBR (hazard ratio, 0.39; P = 0.04) were prognostic biomarkers for TDT irrespective of treatment, and baseline TBR predicted metformin-specific treatment effects that were dependent on baseline tumor hypoxia. Conclusion: Using 18F-flortanidazole PET imaging in a non-small cell lung cancer xenograft model, we showed that metformin may act as a radiosensitizer by increasing tumor oxygenation and that baseline 18F-flortanidazole shows promise as an imaging biomarker.

Noninvasive Whole-Body Imaging of Phosphatidylethanolamine as a Cell Death Marker Using 99mTc-Duramycin During TNF-Induced SIRS.

Systemic inflammatory response syndrome (SIRS) is an inflammatory state affecting the whole body. It is associated with the presence of pro- and antiinflammatory cytokines in serum, including tumor necrosis factor (TNF). TNF has multiple effects and leads to cytokine production, leukocyte infiltration, and blood pressure reduction and coagulation, thereby contributing to tissue damage and organ failure. A sterile mouse model of sepsis, TNF-induced SIRS, was used to visualize the temporal and spatial distribution of damage in susceptible tissues during SIRS. For this, a radiopharmaceutical agent, 99mTc-duramycin, that binds to exposed phosphatidylethanolamine on dying cells was longitudinally visualized using SPECT/CT imaging. Methods: C57BL/6J mice were challenged with intravenous injections of murine TNF or vehicle, and necrostatin-1 was used to interfere with cell death. Two hours after vehicle or TNF treatment, mice received 99mTc-duramycin intravenously (35.44 ± 3.80 MBq). Static whole-body 99mTc-duramycin SPECT/CT imaging was performed 2, 4, and 6 h after tracer injection. Tracer uptake in different organs was quantified by volume-of-interest analysis using PMOD software and expressed as SUVmean After the last scan, ex vivo biodistribution was performed to validate the SPECT imaging data. Lastly, terminal deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was performed to correlate the obtained results to cell death. Results: An increased 99mTc-duramycin uptake was detected in mice injected with TNF, when compared with control mice, in lungs (0.55 ± 0.1 vs. 0.34 ± 0.05), intestine (0.75 ± 0.13 vs. 0.56 ± 0.1), and liver (1.03 ± 0.14 vs. 0.64 ± 0.04) 4 h after TNF and remained significantly elevated until 8 h after TNF. The imaging results were consistent with ex vivo γ-counting results. Significantly increased levels of tissue damage were detected via TUNEL staining in the lungs and intestine of mice injected with TNF. Interestingly, necrostatin-1 pretreatment conferred protection against lethal SIRS and reduced the 99mTc-duramycin uptake in the lungs 8 h after TNF (SUV, 0.32 ± 0.1 vs. 0.51 ± 0.15). Conclusion: This study demonstrated that noninvasive 99mTc-duramycin SPECT imaging can be used to characterize temporal and spatial kinetics of injury and cell death in susceptible tissues during TNF-induced SIRS, making it useful for global, whole-body assessment of tissue damage during diseases associated with inflammation and injury.

[99mTc]duramycin for cell death imaging: Impact of kit formulation, purification and species difference.

INTRODUCTION: [99mTc]duramycin is a SPECT tracer for cell death imaging. We evaluated the impact of kit formulation, purification and species difference on the pharmacokinetic profile and cell death targeting properties of [99mTc]duramycin in order to define the optimal conditions for (pre-)clinical use. METHODS: Three kits were prepared (A: traditional formulation, B: containing 1/3 of ingredients, C: containing HYNIC-PEG12-duramycin). Following labeling, the kits were used without purification, or with SPE or HPLC purification. The pharmacokinetic profile was evaluated in mice and rats at 24 h post tracer injection (p.i.). Non-specific accumulation of [99mTc]duramcyin was studied by µSPECT imaging in chemotherapy treated COLO205 tumor bearing mice pre-treated with cold duramycin (0.01-50 µg). Cell death targeting ability of the kits displaying the best pharmacokinetic profile was compared in a treatment response study in COLO205 tumor bearing mice treated with conatumumab (anti-DR5 antibody). RESULTS: HPLC purification of kit prepared [99mTc]duramycin and reducing the amount of kit ingredients resulted in the best pharmacokinetic profile with low accumulation in liver, spleen and kidneys. The use of PEGylated [99mTc]duramycin required longer circulation times (> 4 h pi) to obtain good imaging characteristics. Pre-treatment with duramycin significantly decreased tracer uptake in chemotherapy treated tumors in a dose-dependent manner. A blocking dose of 50 µg significantly increased non-specific accumulation in liver and spleen. Non-specific accumulation of [99mTc]duramycin was however demonstrated to be species dependent. HPLC purified kit A (5.21±1.71 %ID/cc) and non-purified kit B (1.68±0.46 %ID/cc) demonstrated a significant increase in tumor uptake compared to baseline following conatumumab treatment. CONCLUSIONS: To obtain [99mTc]duramycin with favorable imaging characteristics for cell death imaging in mice [99mTc]duramycin needs to be prepared with high specific activity by applying HPLC purification. The need for HPLC purification appears to be a species dependent phenomenon and might therefore not be required for clinical translation.

Phosphatidylethanolamine targeting for cell death imaging in early treatment response evaluation and disease diagnosis.

Phosphatidylethanolamine (PE) is one of the most abundant phospholipids in mammalian plasma membranes. In healthy cells, PE resides predominantly in the inner leaflet of the cell membrane. In dead or dying cells on the other hand, PE is externalized to the outer leaflet of the plasma membrane. The exposure of PE on the cell surface has therefore become an attractive target for the molecular imaging of cell death using single-photon emission computed tomography (SPECT) and positron emission tomography (PET). This has motivated the development of PE-specific probes to measure cell death in vitro and non-invasively in vivo. In this review, we highlight the biological roles of PE on cell membranes, and PE exposure as a biomarker of cell death in disease processes, along with the use of PE-binding molecular probes to target PE for the characterization of cell death on a cellular and tissue level. We specifically emphasize the preclinical applications of radiolabeled duramycin for the non-invasive imaging of cell death in animal models of disease and in tumors after therapy. In addition, we discuss the clinical relevance, limitations and future perspectives of this imaging approach of cell death.

Evaluation of [18F]CP18 as a Substrate-Based Apoptosis Imaging Agent for the Assessment of Early Treatment Response in Oncology.

PURPOSE: The substrate-based positron emission tomography (PET) tracer [18F]CP18 is capable of detecting the activity of caspase-3/7, two key executioner proteases in the apoptosis pathway, through selective cleavage of the ligand by the activated proteases and subsequent accumulation in apoptotic cells. Using an in vitro and in vivo model of colorectal cancer (CRC), we investigated whether [18F]CP18 tracer accumulation provides a measure for apoptosis and reliably reflects early treatment response to chemotherapeutics. PROCEDURES: [18F]CP18 cell uptake was assessed in treated Colo205 cells (saline, 5-fluorouracil (5-FU), irinotecan or their combination) and correlated with caspase-3/7 activity. [18F]CP18 imaging was performed in Colo205 xenografts, starting with a baseline µPET/micro X-ray computed tomography (​µCT) scan, followed by a 3-day treatment with saline (n = 5), 5-FU (low sensitivity, n = 4), irinotecan (high sensitivity, n = 5), or a combination of both (n = 7). The study was concluded with a second [18F]CP18 scan, 24 h after final treatment administration, followed by tumor removal for gamma counting (%ID/g) and for cleaved caspase-3 immunohistochemistry (apoptotic index/necrosis). Tumors were delineated on µCT images and, using the obtained volumes of interest, average percentage injected dose per cubic centimeter (%ID/cm3) was calculated from every µPET image. RESULTS: In vitro, [18F]CP18 cell uptake was positively correlated with caspase-3/7 activity (r = 0.59, p = 0.003). A drug-dependent increase in [18F]CP18 tumor uptake compared to baseline was observed in animals treated with 5-FU (+14 ± 25 %), irinotecan (+56 ± 54 %), and their combination (+158 ± 69 %, p = 0.002). %ID/cm3 showed a positive relationship with both %ID/g (r = 0.83, p < 0.0001) and the apoptotic index (r = 0.60, p = 0.004), but not with tumor necrosis (r = 0.22, p = 0.36). CONCLUSION: Both our in vitro and in vivo findings have shown the ability of [18F]CP18-PET to visualize therapy-induced cancer cell apoptosis and possibly serve as a biomarker for early therapy response.

99mTc-Duramycin SPECT Imaging of Early Tumor Response to Targeted Therapy: A Comparison with 18F-FDG PET.

Molecular imaging of cell death may provide a detailed readout of the cellular response to novel therapies and prognostic information on tumor treatment efficacy, assisting in the design of individualized therapy. We compared the predictive power of cell death imaging using 99mTc-duramycin with the current gold standard 18F-FDG for treatment response evaluation after targeted therapy. Methods: Early therapy response evaluation was assessed by 99mTc-duramycin SPECT and 18F-FDG PET imaging in treatment-sensitive COLO205 and treatment-resistant HT29 human colorectal cancer xenografts 24 h after a single dose of conatumumab or IgG1 control. The specificity of 99mTc-duramycin for apoptosis was assessed using 99mTc-linear duramycin control radiotracer. Radiotracer uptake was validated ex vivo by γ-counting and autoradiography and compared with cleaved caspase-3 (CC3) activation and DNA fragmentation (TdT-mediated dUTP nick-end labeling [TUNEL]). Data were analyzed with the Student t test and Pearson correlation. All statistical tests were 2-sided. Results: COLO205 tumor uptake of 99mTc-duramycin was increased 7-fold from baseline in conatumumab- versus IgG1-treated control mice (P < 0.001), in good correlation with histologic analysis of apoptosis (CC3, r = 0.842, and TUNEL, r = 0.894; P < 0.001). No response was detected in HT29 tumors. No change in 99mTc-linear duramycin uptake could be detected in COLO205 tumors after treatment, indicating specificity of the 99mTc-duramycin tumor signal. 18F-FDG uptake was not significantly increased from baseline in conatumumab- versus IgG1-treated COLO205 and HT29 tumor-bearing mice (P = 0.104 and 0.779, respectively) and did not correlate with immunohistochemical evidence of apoptosis. Conclusion: We have demonstrated that 99mTc-duramycin specifically accumulates in apoptotic tumors in which 18F-FDG was not able to differentiate responding from nonresponding tumors early after treatment. 99mTc-duramycin holds promise as a noninvasive imaging radiotracer for early treatment evaluation in the clinic.

Evaluation of [18 F]BR420 and [18 F]BR351 as radiotracers for MMP-9 imaging in colorectal cancer.

MMP-9 is a zinc-dependent endopeptidase that is involved in the proteolytic degradation of the extracellular matrix and plays an important role in cancer migration, invasion, and metastasis. The aim of this study was to evaluate the potential of MMP-tracers [18 F]BR420 and [18 F]BR351 for MMP-9 imaging in a colorectal cancer xenograft model. [18 F]BR420 and [18 F]BR351 were synthesized using an automated synthesis module. For [18 F]BR420, a novel and improved radiosynthesis was developed. Plasma stability and MMP-9-targeting capacity of both radiotracers was compared in the Colo205 colorectal cancer model. MMP-9 and MMP-2 expression levels in the tumors were evaluated by immunohistochemistry and in situ zymography. µPET imaging as well as ex vivo biodistribution revealed a higher tumor uptake for [18 F]BR420 (3.15% ± 0.03% ID/g vs 0.94% ± 0.18% ID/g for [18 F]BR351 at 2 hours pi) but slower blood clearance compared with [18 F]BR351. [18 F]BR351 was quickly metabolized in plasma with 20.28% ± 5.41% of intact tracer remaining at 15 minutes postinjection (PI). By contrast, [18 F]BR420 displayed a higher metabolic stability with >86% intact tracer remaining at 2 hours PI. Immunohistochemistry revealed the presence of MMP-9 and MMP-2 in the tumor tissue, which was confirmed by in situ zymography. However, an autoradiography analysis of tracer distribution in the tumors did not correlate with MMP-9 expression. [18 F]BR420 displayed a higher tumor uptake and higher stability compared with [18 F]BR351 but a low tumor-to-blood ratio and discrepancy between tracer distribution and MMP-9 immunohistochemistry. Therefore, both tracers will not be usefulness for MMP-9 imaging in colorectal cancer.

Longitudinal Characterization of [18F]-FDG and [18F]-AV45 Uptake in the Double Transgenic TASTPM Mouse Model.

We aimed to monitor the timing of amyloid-ß deposition in relation to changes in brain function using in vivo imaging with [18F]-AV45 and [18F]-FDG in a mouse model of Alzheimer's disease. TASTPM transgenic mice and wild-type controls were scanned longitudinally with [18F]-AV45 and [18F]-FDG before (3 months of age) and at multiple time points after the onset of amyloid deposition (6, 9, 12, and 15 months of age). As expected with increasing amyloidosis, TASTPM mice demonstrated progressive age-dependent increases in [18F]-AV45 uptake that were significantly higher than for WT from 9 months onwards and correlated to ex vivo measures of amyloid burden. The metabolism of [18F]-AV45 produces several brain penetrant radiometabolites and normalization to a reference region helps to negate this non-specific binding and improve the sensitivity of [18F]-AV45. The observed trajectory of [18F]-FDG alterations deviated from our proposed hypothesis of gradual decreases with worsening amyloidosis. While [18F]-FDG uptake in TASTPM mice was significantly lower than that of WT at 9 months, reduced [18F]-FDG was not associated with aging in TASTPM mice. Moreover, [18F]-FDG uptake did not correlate to measures of ex vivo amyloid burden. Our findings suggest that while amyloid-ß is sufficient to induce hypometabolism, these pathologies are not linked in a dose-dependent manner in TASTPM mice.

Preclinical evaluation of [111 In]MICA-401, an activity-based probe for SPECT imaging of in vivo uPA activity.

Urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 are key players in cancer invasion and metastasis. Both uPA and PAI-1 have been described as prognostic biomarkers; however, non-invasive methods measuring uPA activity are lacking. We developed an indium-111 (111 In)-labelled activity-based probe to image uPA activity in vivo by single photon emission computed tomography (SPECT). A DOTA-conjugated uPA inhibitor was synthesized and radiolabelled with 111 In ([111 In]MICA-401), together with its inactive, hydrolysed form ([111 In]MICA-402). A biodistribution study was performed in mice (healthy and tumour-bearing), and tumour-targeting properties were evaluated in two different cancer xenografts (MDA-MB-231 and HT29) with respectively high and low levels of uPA expression in vitro, with either the active or hydrolysed radiotracer. MicroSPECT was performed 95 h post injection followed by ex vivo biodistribution. Tumour uptake was correlated with human and murine uPA expression determined by ELISA and immunohistochemistry (IHC). Biodistribution data with the hydrolysed probe [111 In]MICA-402 showed almost complete clearance 95 h post injection. The ex vivo biodistribution and SPECT data with [111 In]MICA-401 demonstrated similar tumour uptakes in the two models: ex vivo 5.68 ± 1.41%ID/g versus 5.43 ± 1.29%ID/g and in vivo 4.33 ± 0.80 versus 4.86 ± 1.18 for MDA-MB-231 and HT-29 respectively. Human uPA ELISA and IHC showed significantly higher uPA expression in the MDA-MB-231 tumours, while mouse uPA staining revealed similar staining intensities of the two tumours. Our data demonstrate non-invasive imaging of uPA activity in vivo, although the moderate tumour uptake and hence potential clinical translation of the radiotracer warrants further investigation. Copyright © 2016 John Wiley & Sons, Ltd.

µPET imaging of the pharmacokinetic behavior of medium and high molar mass (89)Zr-labeled poly(2-ethyl-2-oxazoline) in comparison to poly(ethylene glycol).

Poly(2-oxazoline)s are a promising class of polymers for biomedical applications and a versatile alternative to poly(ethylene glycol)s (PEG). In this work, the pharmacokinetic behavior of well defined (89)Zr-labeled poly(2-ethyl-2-oxazoline)s (PEtOx) was evaluated and compared to that of (89)Zr-labeled PEG, both with varying molar mass. Amine-terminated PEtOx of low dispersity in a molar mass range of 20 to 110kDa and PEG of 20 and 40kDa were functionalized with a desferrioxamine chelator and radiolabeled with (89)Zr. The tissue distribution of both radiolabeled PEtOx and PEG polymers was studied by means of micro Positron Emission Tomography (µPET) molecular imaging in mice longitudinally up to 1week post injection, followed by ex vivo biodistribution. As previously described for other classes of non-ionic polymers, the blood clearance of PEtOx decreased with molar mass. The cut off for glomerular filtration of PEtOx is likely to be around 40kDa. The head to head comparison of PEG and PEtOx revealed that the biodistribution is mostly dominated by polymer chain length and not polymer molar mass. This study constitutes an important addition to further establishing PEtOx as a promising polymer in biomedical applications.

Early Prediction of Tumor Response to Treatment: Preclinical Validation of 99mTc-Duramycin.

UNLABELLED: Noninvasive imaging of cell death can provide an early indication of the efficacy of tumor treatment, aiding clinicians in distinguishing responding patients from nonresponding patients early on. (99m)Tc-duramycin is a SPECT tracer for cell death imaging. In this study, our aim was to validate the use of (99m)Tc-duramycin for imaging the early response of tumors to treatment. METHODS: An in vitro binding assay was performed on COLO205 cells treated with 5-fluorouracil (3.1, 31, or 310 µM) and oxaliplatin (0.7 or 7 µM) or radiation (2 or 4.5 Gy). (99m)Tc-duramycin cell binding and the levels of cell death were evaluated after treatment. In vivo imaging was performed on 4 groups of CD1-deficient mice bearing COLO205 human colorectal cancer tumors. Each group included 6 tumors. The first group was given irinotecan (100 mg/kg), the second oxaliplatin (5 mg/kg), the third irinotecan (80 mg/kg) plus oxaliplatin (5 mg/kg), and the fourth vehicle (0.9% NaCl and 5% glucose). For radiotherapy studies, COLO205 tumors received 4.5 Gy, 2 fractions of 4.5 Gy in a 24-h interval, pretreatment with an 80 mg/kg dose of irinotecan combined with 2 fractions of 4.5 Gy in a 24-h interval, or no treatment (n = 5-6/group). Therapy response was evaluated by (99m)Tc-duramycin SPECT 24 h after the last dose of therapy. Blocking was used to confirm tracer specificity. Radiotracer uptake in the tumors was validated ex vivo using γ-counting, cleaved caspase-3, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) histology. RESULTS: Chemotherapy and radiotherapy increased (99m)Tc-duramycin binding to COLO205 cells in a concentration/dose- and time-dependent manner, which correlated well with cell death levels (P < 0.05) as analyzed by annexin V and caspase 3/7 activity. In vivo, (99m)Tc-duramycin uptake in COLO205 xenografts was increased 2.3- and 2.8-fold (P < 0.001) in mice treated with irinotecan and combination therapy, respectively. Blocking with unlabeled duramycin demonstrated specific binding of the radiotracer. After tumor irradiation with 4.5 Gy, (99m)Tc-duramycin uptake in tumors increased significantly (1.24 ± 0.07 vs. 0.57 ± 0.08 percentage injected dose per gram in the unirradiated tumors; P < 0.001). γ-counting of radioactivity in the tumors positively correlated with cleaved caspase-3 (r = 0.85, P < 0.001) and TUNEL (r = 0.81, P < 0.001) staining. CONCLUSION: We demonstrated that (99m)Tc-duramycin can be used to image induction of cell death early after chemotherapy and radiotherapy. It holds potential to be translated into clinical use for early assessment of treatment response.

Baseline [(18)F]FMISO µPET as a Predictive Biomarker for Response to HIF-1α Inhibition Combined with 5-FU Chemotherapy in a Human Colorectal Cancer Xenograft Model.

PURPOSE: The purpose of this study was to characterize imaging biomarkers for the potential benefit of hypoxia-inducible factor-1 (HIF-1)α inhibition (by PX-12) during 5-fluorouracil (5-FU) chemotherapy in the treatment of colorectal cancer (CRC). PROCEDURES: Therapy response to 5-FU ± PX-12 was assessed with baseline [(18)F]fluoromisonidazole ([(18)F]FMISO) and longitudinal 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) positron emission computed tomography (µPET/CT) in CRC xenograft model (n = 36) during breathing of a hypoxic (10 % O2) or normoxic (21 % O2) atmosphere. Ex vivo, immunohistochemistry was performed. RESULTS: Baseline [(18)F]FMISO uptake and relative tumor volume (RTV) 2 days after 5-FU or 5-FU + PX-12 administration correlated significantly (p ≤ 0.01). Under hypoxic breathing conditions, [(18)F]FDG uptake (-53.1 ± 8.4 %) and Ki67 expression (-16 %) decreased and RTV stagnated in the 5-FU + PX-12 treatment group, but not in 5-FU alone-treated tumors. Under normoxic breathing, [(18)F]FDG uptake (-23.5 ± 15.2 % and -72.8 ± 7.1 %) and Ki67 expression (-5 % and -19 %) decreased and RTV stagnated in both the 5-FU and the combination treatment group, respectively. CONCLUSION: Baseline [(18)F]FMISO µPET may predict the beneficial effect of HIF-1α inhibition during 5-FU chemotherapy in CRC.

Multiprobe molecular imaging of an NMDA receptor hypofunction rat model for glutamatergic dysfunction.

There are many indications of a connection between abnormal glutamate transmission through N-methyl-d-aspartate (NMDA) receptor hypofunction and the occurrence of schizophrenia. The importance of metabotropic glutamate receptor subtype 5 (mGluR5) became generally recognized due to its physical link through anchor proteins with NMDAR. Neuroinflammation as well as the kynurenine (tryptophan catabolite; TRYCAT) pathway are equally considered as major contributors to the pathology. We aimed to investigate this interplay between glutamate release, neuronal activation and inflammatory markers, by using small-animal positron emission tomography (PET) in a rat model known to induce schizophrenia-like symptoms. Daily intraperitoneal injection of MK801 or saline were administered to induce the model together with N-Acetyl-cysteine (NAc) or saline as the treatment in 24 male Sprague Dawley rats for one month. Biweekly in vivo [(11)C]-ABP688 microPET was performed together with mGluR5 immunohistochemistry. Simultaneously, weekly in vivo [(18)F]-FDG microPET imaging data for glucose metabolism was acquired and microglial activation was investigated with biweekly in vivo [(18)F]-PBR111 scans versus OX42 immunohistochemistry. Finally, plasma samples were analyzed for TRYCAT metabolites. We show that chronic MK801 administration (and thus elevated endogenous glutamate) causes significant tissue loss in rat brain, enhances neuroinflammatory pathways and may upregulate mGluR5 expression.